Genetic and Biochemical Analyses of Germline Development in Drosophila During development germline is originated from the embryonic germ cells. Eventually the primordial germ cells give rise to sperm and eggs and, therefore, are responsible for producing an entire organism in the next generation. The landmarks of the germline are special organelles, which contain factors necessary for germ cell development including Oskar and Tudor. While there is significant knowledge about the mechanisms that determine differentiation of many somatic cells, little is known about genetic factors involved in germ cell formation and specification. Moreover, composition and function of germline organelles are poorly understood. This project focuses on the identification and characterization of new genes and genes'products essential for the assembly of germline organelles and germ cell development in Drosophila. Many Drosophila genes show striking similarity to human genes and many developmental processes follow the same rules in both fruit flies and humans. Therefore, data obtained during the proposed research should be relevant to humans and may provide critical insight into the biological cause of germline diseases including germline cancers and infertility. In addition, the proposed study should result in better understanding of the role that the Tudor protein plays during the assembly of germline organelles. Tudor contains 11 Tudor domains, which have been evolutionary conserved. In particular, mutations in Tudor domain of human SMN protein cause spinal muscular atrophy which is a leading genetic cause of early childhood mortality. The following specific aims are proposed: I. Identification of the Components of Germline Organelles. Tudor-containing protein complexes present in the ovarian germline organelles will be stabilized by chemical cross-linking and immunopurified. Proteins present in the cross-linked complexes will be identified using mass spectrometry. Localization of identified proteins to germline organelles will be verified in immunostaining experiments. In addition, the effect of mutations in genes that code for the identified proteins on germline development will be examined. If, however, this biochemical approach will encounter technical difficulties due to high non-specific protein background, an alternative genetic approach is proposed as follows. II. Genetic Screen to Identify New Genes Acting Upstream of tudor Gene during Assembly of Germline Organelles. This genetic approach will focus on isolation of the mutants that disrupt the localization of Tudor to ovarian germline organelles and should point to components of these organelles. During the screen localization of Tudor which is fused with Green Fluorescent Protein will be monitored in ovaries. The effect of the mutations on the germline development will be determined and mutant genes will be characterized. PUBLIC HEALTH RELEVANCE: The proposed study should enhance our knowledge of germline development and may provide important insight into the origin of defective sperm and eggs that can be responsible for birth defects. In addition, the proposed research may shed light on the cause of human germline disorders including testicular and ovarian cancers and infertility. Furthermore, the proposed experiments will result in a better understanding of Tudor protein's function during development. Tudor contains multiple repeat modules (called Tudor domains), which are found in various organisms including humans. Importantly, genetic changes in Tudor domain of human SMN protein are responsible for spinal muscular atrophy, which is a leading genetic cause of early childhood mortality (1).